Systematic review of electron transfer study in DNA relevant to Parkinson’s disease and scanning tunneling microscopy

Search strategy

For this systematic search, we developed a search strategy to identify relevant literature. The search strategy utilized three databases, which were Scopus, ScienceDirect, and EBSCOhost MEDLINE by creating general variable search syntax with the following keywords in each database: “electron transfer”, “DNA”, “genetic”, “scanning tunneling”, and “Parkinson’s disease”. The selection of these databases is justified by their relevance, the quality of the content and the focus on the interdisciplinary nature of the research topics in chemistry, biology, and medicine. Together, these databases provide a sufficient general basis for exploring the complex relationships between electron transfer in DNA, oxidative stress, and neurodegenerative diseases. Although the initial outcomes would be limited, the strategic choice of these databases is consistent with the goals of the systematic literature review. However, future research efforts could benefit from a broader selection of databases to ensure a more comprehensive exploration of the literature.

In Scopus (https://www.scopus.com/search/form.uri#basic), we inserted the following several search terms to address the scope of the systematic review paper: TITLE-ABS-KEY (“electron transfer” “Parkinson’s disease”), TITLE-ABS-KEY (“electron transfer” “scanning tunneling” DNA) and TITLE-ABS-KEY (genetic “electron transfer” “Parkinson’s disease”). The search results were 109, 22, and 12 documents, respectively. The total 143 documents were then checked for duplicates, reprints, book chapters, and books, in which a total of 20 documents were considered for exclusion (Fig. 1). ScienceDirect (https://www.sciencedirect.com/) database was used as the second database to search for the literature for the same research topic. The search terms were: (DNA with Title, abstract, keywords: “electron transfer” “Parkinson’s disease”). From the search, 13 documents were obtained and from that, books chapters and short communication (n = 3) were totally excluded. For the third database, EBSCOhost MEDLINE (https://www.ebsco.com/) the search terms used were: (“electron transfer” “Parkinson’s disease” DNA). The result was eight documents, all from academic journals, without any exclusion.

Combining three databases search results, a total of 141 documents were obtained. In the early screening step, duplicate records from the three databases were checked and 18 documents were excluded, leaving altogether 123 documents for further exclusions in eligibility and inclusion steps (Fig. 1). Two authors (MHCL, IW) independently assessed the studies for eligibility and selected the full texts based on the inclusion and exclusion criteria. Any doubts or disagreements in the selection of articles were resolved by discussion and arbitration by another author (MFR).

Selection criteria

Article inclusion and exclusion criteria were based on PRISMA (Liberati et al., 2009; Moher et al., 2009). The search focused on mapping the existing literature on the electron transfer study in the field of genetic PD. The search then narrowed to the subject areas of exclusively in DNA. The search span was from the year 1991 to 2021. All articles before 1991 were excluded from the search. Articles were included in the systematic review if they met the following criteria: (a) The article was written in English. (b) The original research article was published in a peer-reviewed journal. (c) The article reported on a study of electron transfer in PD. (d) The article specified the genetic relationship to PD and STM.

Quality assessment

The study is based on original research and review articles. All duplicate studies were thoroughly screened to ensure the quality of the systematic review. The abstracts of the articles were thoroughly scrutinized for genetic or DNA-related issues associated to PD. The following exclusion criterion was to limit the papers published in English. In addition, after filtering out duplicate records, other types of articles were removed from the study, with the exception of original research journals and reviews. We selected the articles after evaluating each article based on the inclusion and exclusion criteria mentioned above. The process of literature selection and exclusion at each stage, as described in the PRISMA statement, is shown in Fig. 1.

Data extraction

In the data extraction phase, 30 articles were selected and the following information was extracted: (a) Study design of the original research and review papers. (b) Techniques used in the research. (c) Samples or models used in the research. (d) Findings of the research.

Reactive oxygen species generated by mitochondria

Mitochondrial dysfunction is recognized as a common cellular basis for countless human diseases, including classical mitochondrial encephalomyopathies, diabetes, deafness, neurodegenerative diseases, and cancer (El-Khoury et al., 2014; Camargo et al., 2018). The mitochondria are the powerhouse of every cell in our body, generating energy through cellular respiration. However, this process also produces reactive oxygen species (ROS), which can damage various parts of the cells, including proteins, lipids, and DNA. Dysfunctional mitochondria can lead to reduced energy production and cell function.

The decreased electron transfer rates in complexes I and IV, together with other mitochondrial activities, are considered to be the main cause of impaired brain mitochondrial function during aging (Navarro & Boveris, 2010; Jiang et al., 2016). An imbalance between ROS production and antioxidant defense leads to oxidative stress, which promotes various pathophysiological conditions such as diabetes, cancer, arthrosclerosis, aging, Alzheimer’s, and Parkinson’s disease (Umeno, Biju & Yoshida, 2017). These oxidative reactions in cells can produce superoxide anions and hydrogen peroxide (H₂O₂) in large quantities. A reduced metal ion, such as Fe²⁺ or Cu⁺, can react with H₂O₂ and produce a highly reactive hydroxyl radical (Fato et al., 2008). While most products of oxidative metabolism are beneficial, an imbalance in ROS homeostasis leads to oxidative stress and triggers disease. In PD, these findings suggest that antioxidant defenses are overwhelmed, leading to increased oxidative stress.

Electron leakage upstream of the rotenone binding site in complex I (Hensley et al., 1998) and impaired distal electron transfer in complex I significantly increase H₂O₂ production. Therefore, mild genetic or acquired complex I abnormalities that are inadequate to affect respiration may result in persistent upregulation of ROS generation (Greenamyre et al., 2001). Neurons, as postmitotic cells that are routinely exposed to high oxygen tensions and calcium levels, may be more vulnerable to the accumulated oxidative stress during their lifetime.

Increased amount of the heme protein cytochrome c in α-synuclein aggregation

Heme is a precursor to hemoglobin, which is an essential protein in red blood cells. Hemoglobin is responsible for the binding of oxygen in the lungs and its transportation to the rest of the body via the bloodstream. The basic structure of the heme consists of Fe²⁺ that can be found in animal-derived foods. Iron in the heme is an intrinsic generator of ROS. Elevated iron levels enhance neurotoxicity through the generation of hydroxyl radicals, leading to glutathione depletion, protein aggregation, lipid peroxidation, and nucleic acid alterations (Núñez et al., 2012).

A heme iron center is essential for the function of cytochrome c, which has two clearly defined physiological functions: regulating electron transfer in mitochondria and mediating apoptotic cell death. Most of cytochrome c in mitochondria is tightly packed in the inner membrane along with other components of the electron transport chain (Jiang et al., 2016). Since cytochrome c plays a crucial role in electron transfer between the III (ubiquinol:cytochrome oxidoreductase) and the IV (cytochrome oxidase) complexes, dysfunction of cytochrome c molecules can lead to the production of ROS in mitochondria, thereby worsening intracellular conditions for oxidative stress (Shigenaga, Hagen & Ames, 1994). When both cytochrome c and H₂O₂ were present, α-synuclein clumped together more easily, forming dimers and insoluble clumps.

α-Synuclein is a presynaptic neuronal protein that is genetically and neuropathologically associated with PD. Abnormal soluble oligomeric conformations of α-synuclein known as protofibrils are the toxic species that cause disruption of cellular homeostasis and neuronal death by affecting a variety of intracellular targets, including synaptic function (Stefanis, 2012). The synaptic effects of α-synuclein overexpression include loss of presynaptic proteins, reduced neurotransmitter release, expansion of synaptic vesicles, and suppression of synaptic vesicle recycling. Such deficits are likely to precede overt neuropathology and contribute to synaptic and neuritic degeneration, but the exact point in the cascade at which α-synuclein assumes its neurotoxic potential remains elusive.

Complex I malfunction in Parkinson’s disease

NADH:ubiquinone oxidoreductase, known as complex I, is located in the inner mitochondrial membrane and extends into the mitochondrial matrix (Greenamyre et al., 2001). It is an important component of energy metabolism, as this is where most of the electrons enter the respiratory chain and it is the largest and most complex element of the respiratory chain.

Abnormalities of mitochondrial complex I have been implicated in severe, infantile, and childhood neurological disorders as well as late-onset neurodegenerative diseases, such as PD. The exact role of complex I in these illnesses remains controversial, and the mechanisms by which complex I defects cause neurodegeneration are not entirely clear (Greenamyre et al., 2001). Since complex I is a critical component of the electron transfer chain that contributes to adenosine triphosphate (ATP) synthesis and mitochondrial maintenance, severe defects in complex I activity can lead to reduced ATP synthesis, graded mitochondrial depolarization, and calcium dysregulation. Mild genetic or acquired defects of complex I can also lead to chronic up-regulation of ROS production and may contribute to chronic oxidative insult in postmitotic cells like neurons. These cumulative effects may predispose neurons to calcium-mediated damage and result in neurological diseases.

Epidemiological and biochemical studies of post-mortem brain specimens and peripheral tissues from PD patients have revealed that minor systemic complex I abnormalities have a role in the etiology of the disease (Greenamyre et al., 2001). Since 2006, the standardized incidence rates of PD are eight to 18 per 100,000 person-years, and prospective population-based studies indicate that the occurrence of PD is infrequent prior to the age of 50, but there is a significant rise in prevalence after the age of 60 (De Lau & Breteler, 2006). Epidemiological studies indicate that 10% of PD cases are strictly familial, while the majority are sporadic (Thomas & Beal, 2007).

PINK1-induced mitochondrial dysfunction

PINK1 is a protein encoded by the PINK1 gene, associated with the genetic causes of PD. The PINK1 gene is located on chromosome 1p35-p36, which is part of the nuclear DNA. Mutations in this gene can cause early-onset PD, specifically an autosomal recessive form of the disorder (Rogaeva et al., 2004). While PINK1 is encoded by nuclear DNA, its primary function and the consequences of its mutations are manifested in the mitochondria. The mitochondrial dysfunction observed in PINK1-related PD is not due to mutations in mitochondrial DNA itself but rather stems from the failure of the mitochondrial quality control mechanisms that PINK1 regulates (Abramov et al., 2011; Chin et al., 2023). This distinction is crucial; it indicates that while the genetic basis of PINK1 mutations originates from nuclear DNA, the pathological effects are predominantly mitochondrial.

As a key regulator of mitochondrial health, the importance of PINK1 lies in its involvement in the identification and removal of damaged mitochondria through autophagic mechanisms (Li et al., 2023). Mutations or deficiencies in PINK1 have been linked to impaired mitophagy, resulting in the accumulation of dysfunctional mitochondria and subsequent neurodegeneration observed in PD. Temelie, Savu & Moisoi (2018) proposed a model that PINK1 and Parkin constitute the main system for sensing and modulating removal of dysfunctional mitochondria. Under basal conditions in healthy mitochondria, PINK1 is completely imported into the mitochondrial matrix, where it is immediately destroyed by proteolysis (Yamano & Youle, 2013; Temelie, Savu & Moisoi, 2018). Recessive mutations of the PINK1 gene are the second most common factor in causing autosomal recessive early-onset PD, which is an incurable neurodegenerative movement disorder (Lill, 2016). PINK1-Parkin signaling regulates axonal mitochondrial redistribution in human dopaminergic neurons in response to reduced mitochondrial membrane potential, which is impaired in patients with PINK1 and Parkin mutations (Imai, 2020). However, a previously uncharacterized function for the mitochondrial inner membrane lipid cardiolipin increment has been found to rescue PINK1-induced mitochondrial dysfunction (Vos et al., 2017).

Scanning tunneling microscopy in DNA of Parkinson’s disease

STM is a useful technique for studying the detailed properties of material surfaces at the nanoscale. This technique is based on the phenomenon of quantum tunneling in the field of quantum mechanics. In tunneling, electrons are emitted from a sample when a thin sharp probe is brought very close to the sample surface. The tunneling current is generated when the electrons cross a potential barrier and reach the tip of the probe. To ensure a uniform tunneling current, the distance between the tip and the sample surface is adjusted so that the probe maintains this distance precisely while scanning the surface synchronously. This scanning allows the STM to achieve atomic resolution, although it is mainly suitable for conductive materials. Rodríguez-Galván & Contreras-Torres (2022) demonstrate that the STM can be used to observe biological molecules, particularly in the study of double-stranded DNA, which was the first biomolecule to be studied with the STM. This ability to provide detailed spatial information about surface-bound biomolecules is particularly advantageous when studying the intricate structures of DNA and its interactions with electrons, thus facilitating the investigation of DNA damage at the molecular level. In contrast, conventional methods such as fluorescence and electron microscopy to study specific molecular interactions and dynamics may not provide the same level of detail in terms of electronic states and charge transport mechanisms (Maeda, Matsumoto & Kawai, 2011; Elliott & Jones, 2018).

A novel approach to the study of PD is proposed, focusing on electron transport through DNA using microscopic research tools. Electron transfer is one of the fundamental keys to understanding the biological functions of damage repair mechanisms in DNA. In physics and chemistry, a number of experimental studies have generally focused on the electron transport properties of the materials under investigation. As STM is particularly well suited to the study of complex interactions between DNA and other biomolecules or nanoparticles that can affect electron transfer rates, the technique enables the investigation of molecular assemblies and the effects of external perturbations, such as electric fields or chemical modifications, on electron transport (Elliott & Jones, 2018; Fereiro et al., 2018). This capability is less accessible using conventional techniques, which may not provide the same level of insight into the dynamic interactions that occur in biological systems. The development of electrochemical STM further enhances the applicability of STM in biological research by enabling the study of electron transfer processes in situ under conditions that mimic physiological environments (Albrecht, 2012; Cucinotta, Rungger & Sanvito, 2012).

To our knowledge, despite numerous research using various microscopic techniques, including STM, to study electron transfer, there have been few studies using this particular technique to investigate the dynamics of electron transfer in abnormal or damaged DNA in PD. Our novel proposal for DNA research thus builds a bridge between physics and medical research. We propose to use STM as one of the microscopic tools, where STM measurement would provide us with information on electron transfer and authentic structural images at the nanometer scale in the DNA of PINK1 in PD. The microscopic properties of the DNA molecules measured by STM would provide invaluable information about the height profile, which indirectly corresponds to the tunneling current and provides topographical data with more precise measurements than the transmission electron microscope (Rafati & Gill, 2016).

By performing these experiments on normal and damaged DNA molecule samples in PD, we could investigate how electron transfer is affected by damage in DNA. The measurements of the height profile and the current–voltage (I–V) curve are essential for determining and explaining the differences in electron transfer between DNA molecules in PD. From the images of the DNA in PD measured with the STM, the brightness of the images on the DNA molecules can be used to determine how strongly the DNA molecules are electrically conductive. The difference in the brightness of the images will also help us to qualitatively recognize whether the differences in the DNA sequences can be identified by comparing the structures of the DNA. On the other hand, the I–V curves measured on the DNA molecules would give us information about the conductivity information of the molecules, i.e., whether the molecules have the properties of an insulator or electrical conductor. The differential conductance (dI/dV–V) curve patterns, particularly the peak positions in such curves, could serve as a discrete energy spectrum of DNA molecules and be characterized as a fingerprint (Shapir et al., 2010).